Download Lab technique RFLP PPT

-GLOBIN MUTATIONS AND SICKLE CELL
DISORDER (SCD)
- RESTRICTION FRAGMENT LENGTH
POLYMORPHISMS (RFLP)
-GLOBIN MUTATIONS AND SICKLE CELL DISORDER (SCD)
•Well over 700 abnormal forms of haemoglobin have been
identified and characterised to some extent on the basis of the
mutation responsible and/or clinical consequences.
•Over 1000 variants of the globin genes have been identified.
•A variety of mutation mechanisms are responsible for the
abnormal haemoglobins. These mutations affect either
haemoglobin structure or synthesis.
•The most common are point mutations in the –globin gene
resulting in a nucleotide substitution and a change of encoded
amino acid which affects haemoglobin structure.
-GLOBIN POINT MUTATIONS – Hb A, Hb S and HbC
Exon 1
1
2
3
4
5
6
7
8
9 10
atg gtg cat ctg act cct GAG gag aag tct gcc gtt act gcc ctg tgg ggc aag gtg
M
V
H
L
T
P
E
E
K
S
A
V
T
A
L
W
G
K
V
26
aac gtg gat gaa gtt ggt ggt GAG gcc ctg ggc ag
N
V
D
E
V
G
G
E
A
L
G
R
Exon 2
g ctg
L
gat gct gtt
D
A
V
ctg gct cac
L
A
H
gtg gat cct
V
D
P
Exon 3
ctg
L
atg
M
ctg
L
gag
E
gtg
V
ggc
G
gac
D
aac
N
gtc
V
aac
N
aac
N
ttc
F
tac cct tgg acc cag agg ttc ttt gag tcc ttt ggg gat ctg tcc act cct
Y
P
W
T
Q
R
F
F
E
S
F
G
D
L
S
T
P
cct aag gtg aag gct cat ggc aag aaa gtg ctc ggt gcc ttt agt gat ggc
P
K
V
K
A
H
G
K
K
V
L
G
A
F
S
D
G
ctc aag ggc acc ttt gcc aca ctg agt gag ctg cac tgt gac aag ctg cac
L
k
G
T
F
A
T
L
S
E
L
H
C
D
K
L
H
agg
R
121
ctc ctg ggc aac gtg ctg gtc tgt gtg ctg gcc cat cac ttt ggc aaa GAA ttc
L
L
G
N
V
L
V
C
V
L
A
H
H
F
G
K
E
F
acc cca cca gtg cag gct gcc tat cag aaa gtg gtg gct ggt gtg gct aat gcc ctg gcc
T
P
P
V
Q
A
A
Y
Q
K
V
V
A
G
V
A
N
A
L
A
cac aag tat cac taa
H
K
Y
H
*
POINT MUTATIONS IN THE –GLOBIN GENE AND SCD – Hb S
Codons 1-10 of –globin
A.
1
2
3
4
5
6
7
8
9 10
gtg cat ctg act cct GAG gag aag tct gcc
V
H
L
T
P
E
E
K
S
A
B. Hb A (A allele)
5
6
7
cct GAG gag
P
E
E
The “usual” allele is referred to as the A allele and produces Hb A
C. Hb S (S allele)
cct GTG gag 20A>T mutation
P
V
E
The sickle cell mutation or S allele. In this mutation the 20th nucleotide is
mutated from an “A” to a “T” which causes a change in the amino acid encoded
by this codon from glutamic acid (E) to valine (V)
POINT MUTATIONS IN THE –GLOBIN GENE AND SCD – Hb C
Codons 1-10 of –globin
A.
1
2
3
4
5
6
7
8
9 10
gtg cat ctg act cct GAG gag aag tct gcc
V
H
L
T
P
E
E
K
S
A
B. Hb A (A allele)
5
6
7
cct GAG gag
P
E
E
The “usual” allele is referred to as the A allele and produces Hb A
C. Hb C (C allele)
cct AAG gag 19G>A mutation
P
K
E
The Hb C mutation or C allele. In this mutation the 19th nucleotide mutates from a
“g” to an “a” which causes a change in the amino acid encoded from glutamic acid
(E) to lysine (K)
IDENTIFYING THE Hb S MUTATION USING THE POLYMERASE CHAIN
REACTION (PCR) AND RESTRICTION FRAGMENT LENGTH
POLYMORPHISM (RFLP)
MUTATION ABOLISHES RESTRICTION SITE
AMPLIFY PORTION OF -GLOBIN GENE BY PCR
Firstly a portion of the –globin gene is amplified by the PCR. The region
of DNA amplified must contain the specific DNA sequence that is mutated
and that mutation must introduce or abolish a restriction site.
DIGEST PRODUCTS WITH RESTRICTION ENZYME
The amplified DNA is then digested with the specific restriction
endonuclease. Mutated and non-mutated amplicons will have different
restriction sites in them, giving different patterns of bands on
electrophoresis
ANALYSE PRODUCTS BY GEL ELECTROPHORESIS
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
A.
4 5 6 7 8
actcctGAGgagaag
tgaggaCTCctcttc
B.
MstII = CCTNAGG

C.
actcctGAGgagaag
tgaggaCTCctcttc

MstII
actcc
tGAGgagaag
tgaggaCT
Cctcttc
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
A.
4 5 6 7 8
actcctGAGgagaag
tgaggaCTCctcttc
C.
B.
CTNAG = DdeI

actcctGAGgagaag
tgaggaCTCctcttc

DdeI
actcc
tGAGgagaag
tgaggaCT
Cctcttc
A.
Using DdeI
6
EXON 1
EXON 2
EXON 3
B.
571 bp amplicon
C.
A allele
107bp
201bp
88bp
89bp
49bp
37bp
S allele
308bp
88bp
89bp
49bp
37bp
Gel Electrophoresis
Using MstII
A allele
S allele
223bp
201bp
424bp
88bp
242bp
88bp
242bp
Gel Electrophoresis